Difference between revisions of "Team:SDU-CHINA/Improve"

 
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      <br> <h1 id="main-title">Improve</h1><br><br>
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      <br> <h1 id="main-title">Improve</h1> <br>
 
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   <p><b>Advanced CcaS with PSA domain knocked out with an opposite response to green/red light.</b> <br>
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   <div class="bba"  style="text-align: center;"><h4><b><a href="http://parts.igem.org/Part:BBa_K2627000">BBa_K2627000</a></b></h4></div>
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CcaS is a membrane protein functioning in CcaS/CcaR two-component system in response to green/red light. Under green light, the system and activate the transcription and expression of the specific genes downstream a proper promoter named PcpcG2, while the expression of the genes can be repressed when exposed to red light. This year, we were inspired by Professor Sode’ work of making a modification of CcaS’s structure and functions. And we deleted 858 base pairs in gene coding for CcaS, which means we removed the PSA domain in CcaS protein. When we characterized this new part, we surprised to find that the new CcaS had an opposite response to green/red light, compared to the original one.
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<p>&nbsp;&nbsp;&nbsp;&nbsp;This year we made an improvement on part <a href="http://parts.igem.org/Part:BBa_K592001">BBa_K592001</a> , which codes for green/red light sensing protein CcaS. CcaS functions in CcaS-CcaR two-component system responding to the existence of green or red light. CcaS consists of the following domains and regions: a N-terminal transmembrane helix, a sensor domain consisting of a cyanobacteriochrome-type cyclic guanosine monophosphate phosphodiesterase/adenylyl cyclase/formate hydrogen lyase transcriptional activator (GAF) domain, a linker region, two period/aryl hydrocarbon receptor nuclear translocator/single-minded (PAS) domains of unknown function, a second linker region, and a C-terminal HK domain (Fig. 1) <a href="#r1"><sup>[1]</sup></a>. Previously, the functions of some of the domains were studied, for example, a shape change can be observed in the GFA domain after CcaS was exposed to light, which could thus induce an autophosphorylation of the HK domain, then the phosphate group would be transferred to the regulator protein CcaR<a href="#r2"><sup>[2]</sup></a>. However, the function of PSA domains is remained unknown.
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<br><a href="http://parts.igem.org/Part:BBa_K2627000">http://parts.igem.org/Part:BBa_K2627000</a> <br></p>
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<img src="https://static.igem.org/mediawiki/2018/e/e2/T--SDU-China--chara.png" alt="">
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<img src="https://static.igem.org/mediawiki/2018/8/85/T--SDU-China--improve.png" alt=""> <br>
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<p class="reference"><b>Figure 1.</b> A brief illustration of the structure of CcaS. TF domain is located in the N-terminal while HK domain shows where the C-terminal is.</p>
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  <br>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;We were inspired by Professor Koji’s work[1] in which he removed the two PSA domains to see what would happen. In his work, he created 11 variants of CcaS by deleting different numbers of base pairs, and these variants were characterized through measuring fluorescence/OD. We repeated his work and acquired this new part. This improved part is #4 in the variants collection. We found that this new part has a totally opposite response to green and red light compared with the original CcaS. As we can see from the figure below, #4 has a high fluorescence/Abs600 under red light but has a low one under green light.</p>
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<img src="https://static.igem.org/mediawiki/2018/2/26/T--SDU-China--improve2.png" alt=""> <br>
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<p class="reference"><b>Figure 2. </b>Characterization of part BBa_K2627000 and BBa_K592001. Part BBa_K2627000 shows a different effect after induced by green light and red light. Then fluorescence intensity of CcaS#4 was measured at 9h.</p>
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<br><br>
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<h4>References</h4>
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<p class="reference">
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[1].    Nakajima, M., et al., Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal. Scientific Reports, 2016. 6(1).
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<br>
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[2].  Hirose, Y., Shimada, T., Narikawa, R., Katayama, M. & Ikeuchi M. Cyanobacteriochrome CcaS is the green light receptor that
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induces the expression of phycobilisome linker protein. Proc. Natl. Acad. Sci. USA 105, 9528–9533 (2008).
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</p>
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Latest revision as of 03:58, 18 October 2018


Improve


BBa_K2627000

    This year we made an improvement on part BBa_K592001 , which codes for green/red light sensing protein CcaS. CcaS functions in CcaS-CcaR two-component system responding to the existence of green or red light. CcaS consists of the following domains and regions: a N-terminal transmembrane helix, a sensor domain consisting of a cyanobacteriochrome-type cyclic guanosine monophosphate phosphodiesterase/adenylyl cyclase/formate hydrogen lyase transcriptional activator (GAF) domain, a linker region, two period/aryl hydrocarbon receptor nuclear translocator/single-minded (PAS) domains of unknown function, a second linker region, and a C-terminal HK domain (Fig. 1) [1]. Previously, the functions of some of the domains were studied, for example, a shape change can be observed in the GFA domain after CcaS was exposed to light, which could thus induce an autophosphorylation of the HK domain, then the phosphate group would be transferred to the regulator protein CcaR[2]. However, the function of PSA domains is remained unknown.


Figure 1. A brief illustration of the structure of CcaS. TF domain is located in the N-terminal while HK domain shows where the C-terminal is.


    We were inspired by Professor Koji’s work[1] in which he removed the two PSA domains to see what would happen. In his work, he created 11 variants of CcaS by deleting different numbers of base pairs, and these variants were characterized through measuring fluorescence/OD. We repeated his work and acquired this new part. This improved part is #4 in the variants collection. We found that this new part has a totally opposite response to green and red light compared with the original CcaS. As we can see from the figure below, #4 has a high fluorescence/Abs600 under red light but has a low one under green light.


Figure 2. Characterization of part BBa_K2627000 and BBa_K592001. Part BBa_K2627000 shows a different effect after induced by green light and red light. Then fluorescence intensity of CcaS#4 was measured at 9h.



References

[1]. Nakajima, M., et al., Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal. Scientific Reports, 2016. 6(1).
[2]. Hirose, Y., Shimada, T., Narikawa, R., Katayama, M. & Ikeuchi M. Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proc. Natl. Acad. Sci. USA 105, 9528–9533 (2008).